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Target

ShK toxin blocks the K+ channels Kv1.1, Kv1.3, Kv1.6, Kv3.2 and KCa3.1,[13][14][15][16][17] The peptide binds to all four subunits in the Kv1.3 tetramer through its interaction with the shallow vestibule at the outer entrance of the ion conduction pathway.[13][14][18] The peptide's Lysine22 residue occludes the channel pore like a "cork in a bottle". This blocks the entrance to the pore.[19][20]

Schematic diagram of the primary structure of the ShK peptide highlighting the three disulfide (–S–S–) linkages.

ShK blocks the Kv1.3 channel in T cells with a Kd of about 11 pM.[13][14][21] It blocks the neuronal Kv1.1 and Kv1.6 channels with Kds of 16 pM and 200 pM respectively.[16] The Kv3.2 and KCa3.1 channels are more than 1000 times less sensitive to the peptide.[13][14][16][17]

Several ShK analogs have been generated to enhance specificity for the Kv1.3 channel over the Kv1.1, Kv1.6 and Kv3.2 channels. The first analog that showed some degree of specificity was ShK-Dap22.[13] Attaching a fluorescein to the N-terminus of the peptide via a hydrophilic AEEA linker (2-aminoethoxy-2-ethoxy acetic acid; mini-PEG) resulted in a peptide, ShK-F6CA, with 100-fold specificity for Kv1.3 over Kv1.1 and related channels.[21] Based on this surprising finding additional analogs were made. ShK-170 [a.k.a. ShK(L5)], contains a L-phosphotyrosine in place of the fluorescein in ShK-F6CA. It blocks Kv1.3 with a Kd of 69 pM and shows exquisite specificity for Kv1.3.[16] However, it is chemically unstable. To improve stability a new analog, ShK-186 [a.k.a. SL5], was made with the C-terminal carboxyl of ShK-170 replaced by an amide; ShK-186 is otherwise identical to ShK-170.[22][23] In rats and squirrel monkeys, an indium-labeled ShK-186 analog called ShK-221, was slowly released from the injection site and maintained blood levels above the channel blocking dose for 3–5 days [24] ShK-192 is a new analog with increased stability.[23] It contains norleucine21 in place of methionine21 to avoid methionine oxidation, and the terminal phosphotyrosine is replaced by a non-hydrolyzable para-phosphonophenylalanine (Ppa) group.[23] ShK-192 is effective in ameliorating disease in rat models of multiple sclerosis. The D-diastereomer of ShK is also stable but blocks Kv1.3 with 2800-fold potency than the L-form (Kd = 36 nM) and it only exhibits 2-fold specificity for Kv1.3 over Kv1.1.[25] ShK-K-amide is a new analog with a C-terminal lysine. It blocks Kv1.3 with roughly 50-fold greater potency (IC50 of 26 ± 3 pM) than Kv1.1 ( IC50 of 942 ± 120 pM), and suppresses proliferation of human T cells (IC50 ≈ 3 nM).[26]

Kv1.3 and KCa3.1 regulate membrane potential and calcium signaling of T cells.[19] Calcium entry through the CRAC channel is promoted by potassium efflux through the Kv1.3 and KCa3.1 potassium channels.[22] Blockade of Kv1.3 channels in effector-memory T cells by ShK-186 suppresses calcium signaling, cytokine production (interferon-gamma, interleukin 2) and cell proliferation.[19][27][22] In vivo, ShK-186 paralyzes effector-memory T cells at the sites of inflammation and prevent their reactivation in inflamed tissues.[28] In contrast, ShK-186 does not affect the homing to and motility within lymph nodes of naive and central memory T cells, most likely because these cells express the KCa3.1 channel and are therefore protected from the effect of Kv1.3 blockade.[28] In proof-of-concept studies, ShK and its analogs have prevented and treated disease in rat models of multiple sclerosis, rheumatoid arthritis, and delayed type hypersensitivity.[21][29][16][29] ShK-186, due to its durable pharmacological action, is effective in ameliorating disease in rat models of delayed type hypersensitivity, multiple sclerosis (experimental autoimmune encephalomyelitis) and rheumatoid arthritis (pristane induced arthritis) when administered once every 2–5 days.[24] ShK-186 has completed non-clinical safety studies. ShK-186 is the subject of an open Investigational New Drug (IND) application in the USA, and has completed human phase 1A and 1B trials in healthy volunteers.

As ShK toxin binds to the synaptosomal membranes, it facilitates an acetylcholine release at avian neuromuscular junctions while the Kv3.2 channels are expressed in neurons that fire at a high frequency (such as cortical GABAergic interneurons), due to their fast activation and deactivation rates.[17] By blocking Kv3.2, ShK toxin depolarises the cortical GABAergic interneurons. Kv3.2 is also expressed in pancreaticbeta cells. These cells are thought to play a role in their delayed-rectifier current, which regulates glucose-dependent firing. Therefore, ShK, as a Kv3.2 blocker, might be useful in the treatment of type-2 diabetes, although inhibition of the delayed-rectifier current has not yet been observed in human cells even when very high ShK concentrations were used.[17]

Toxicity

Toxicity of ShK toxin in mice is quite low. The median paralytic dose is about 25 mg/kg bodyweight (which translates to 0.5 mg per 20 g mouse). In rats the therapeutic safety index was greater than 75-fold.

ShK-Dap22 is less toxic, even a dose of 1.0 mg dose did not cause hyperactivity, seizures or mortality. The median paralytic dose was 200 mg/kg body weight.[13]

ShK-170 [a.k.a. ShK(L5)] does not cause significant toxicity in vitro. The peptide was not toxic to human and rat lymphoid cells incubated for 48 h with 100 nM of ShK-170 (>1200 times greater than the Kv1.3 half-blocking dose). The same high concentration of ShK-170 was negative in the Ames test on tester strain TA97A, suggesting that it is not a mutagen. ShK-170 had no effect on heart rate or heart rate variability parameters in either the time or the frequency domain in rats. It does not block the hERG (Kv11.1) channel that is associated with drug-associated cardiac arrhythmias. Repeated daily administration of the peptide by subcutaneous injection (10 µg/kg/day) for 2 weeks to rats does not cause any changes in blood counts, blood chemistry or in the proportion of thymocyte or lymphocyte subsets. Furthermore, the rats administered the peptide gain weight normally.

ShK-186 [a.k.a. SL5] is also safe. Repeated daily administration by subcutaneous injection of ShK-186 (100 µg/kg/day) for 4 weeks to rats does not cause any changes in blood counts, blood chemistry or histopathology.[22] Furthermore, ShK-186 did not compromise the protective immune response to acute influenza viral infection or acute bacterial (Chlamydia) infection in rats at concentrations that were effective in ameliorating autoimmune diseases in rat models.[28] Interestingly, rats repeatedly administered ShK-186 for a month by subcutaneous injection (500 µg/kg/day) developed low titer anti-ShK antibodies.[29] The reason for the low immunogenicity of the peptide is not well understood. ShK-186 has completed GLP (Good Laboratory Practice) non-clinical safety studies in rodents and non-human primates. ShK-186 (aka Dalazatide) which was licensed to Kineta Bio is the subject of an open Investigational New Drug (IND) application in the United States of America, and has recently completed human phase 1A and 1b trials in healthy volunteers. A second human phase 1b was recently completed in 2015 in psoriasis patients. Dalazatide was shown to significantly ameliorate symptoms in 90% patients with active plaque psoriasis with a 60 mcg weekly dose.

Many groups are developing Kv1.3 blockers for the treatment of autoimmune diseases.[30]

Use

Because ShK toxin is a specific inhibitor of Kv1.1, Kv1.3, Kv1.6, Kv3.2 and KCa3.1, it may serve as a useful pharmacological tool for studying these channels.[17][21] The Kv1.3 specific ShK analogs, ShK-170, ShK-186 and ShK-192, have been demonstrated to be effective in rat models of autoimmune diseases, and these or related analogs might have use as therapeutics for human autoimmune diseases.

Kv1.3 is also considered a therapeutic target for the treatment of obesity,[31][32] for enhancing peripheral insulin sensitivity in patients with type-2 diabetes mellitus,[33] and for preventing bone resorption in periodontal disease.[34] Furthermore, because pancreatic beta cells, which have Kv3.2 channels, are thought to play a role in glucose-dependent firing, ShK, as a Kv3.2 blocker, might be useful in the treatment of type-2 diabetes, although inhibition of the delayed-rectifier current has not yet been observed in human cells even when very high ShK concentrations were used.[16]

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This domain of is found in several C. elegans proteins. The domain is 30 amino acids long and rich in cysteine residues. There are 6 conserved cysteine positions in the domain that form three disulphide bridges. The domain is found in the potassium channel inhibitor ShK in sea anemone [1].

External database links

BgK, a 37-residue peptide toxin from the sea anemone Bunodosoma granulifera, and ShK, a 35-residue peptide toxin from the sea anemone Stichodactyla helianthus, are potent inhibitors of K(+) channels. There is a large superfamily of proteins that contains domains (referred to as ShKT domains) ressembling these two toxins. Many of these proteins are metallopeptidases, whereas others are prolyl-4-hydroxylases, tyrosinases, peroxidases, oxidoreductases, or proteins containing epidermal growth factor-like domains, thrombospondin-type repeats, or trypsin-like serine protease domains [PUBMED:19965868]. The ShKT domain has also been called NC6 (nematode six-cysteine) domain [PUBMED:10950959], SXC (six-cysteine) domain [PUBMED:10950959, PUBMED:11412804, PUBMED:9851921, PUBMED:14653817] and ICR (ion channel regulator) [PUBMED:19965868, PUBMED:16339766]. The ShKT domain is short (36 to 42 amino acids), with six conserved cysteines and a number of other conserved residues. The fold adopted by the ShKT domain contains two nearly perpendicular stretches of helices, with no additional canonical secondary structures [PUBMED:9020148]. The globular architecture of the ShKT domain is stabilised by three disulfides, one of them linking the two helices. In venomous creatures, the ShKT domain may have been modified to give rise to potent ion channel blockers, whereas the incorporation of this domain into plant oxidoreductases and prolyl hydroxylases and into worm astacin-like metalloproteases and trypsin-like serines protaeses produced enzymes with potential channel-modulatory activity.

Toxocara canis family of secreted mucins Tc-MUC-1 to -5, which are implicated in immune evasion. They combine two evolutionarily distinct modules, the mucin and ShkT domains [PUBMED:10950959, PUBMED:11412804].

Domain organisation

Below is a listing of the unique domain organisations or architectures in which
this domain is found.
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The graphic that is shown by default represents the longest sequence
with a given architecture. Each row contains the following information:

the number of sequences which exhibit this architecture

a textual description of the architecture, e.g. Gla, EGF x 2, Trypsin.
This example describes an architecture with one Gla
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finally a single Trypsin domain

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Note that you can see the family page for a particular domain by
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Pfam Clan

This family is a member of clan ShK-like
(CL0213),
which has the following description:

Members of this clan include the Crisp domain which is involved in ryanodine receptor Ca2+ signalling, and the ShK domain which is named after the ShK channel inhibitor toxin. Both domains are cysteine rich and contain multiple disulphide bonds [1][2][3].

Alignments

We store a range of different sequence alignments for families. As well
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Seed(140)

Full(7196)

Representative proteomes

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NCBI(7189)

Meta(124)

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1

1Cannot generate PP/Heatmap alignments for seeds; no PP data available

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Format an alignment

Seed(140)

Full(7196)

Representative proteomes

UniProt(8908)

NCBI(7189)

Meta(124)

RP15(3479)

RP35(4681)

RP55(6212)

RP75(6604)

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Trees

This page displays the phylogenetic tree for this family's seed
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How the sunburst is generated

The tree is built by considering the taxonomic lineage of each
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The tree is built by looking at each sequence in the full
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any node in the NCBI tree, perhaps because the chosen name is
listed as a synonym or a misspelling in the NCBI taxonomy.

So that these nodes are not simply omitted from the sunburst
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superkingdom and the species as "uncategorised".

Sub-species

Since we reduce the species tree to only the eight main
taxonomic levels, sequences that are mapped to the sub-species
level in the tree would not normally be shown. Rather than leave
out these species, we map them instead to their parent species.
So, for example, for sequences belonging to one of the
Vibrio cholerae sub-species in the NCBI taxonomy, we
show them instead as belonging to the species Vibrio
cholerae.

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The tree shows the occurrence of this domain across different species.
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Species trees

We show the species tree in one of two ways. For smaller trees we try
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Unfortunately we have found that there are problems viewing the
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Interactive tree

For all of the domain matches in a full alignment, we count the
number that are found on all sequences in the alignment.
This total is shown in the purple box.

We also count the number of unique sequences on which each domain is
found, which is shown in green.
Note that a domain may appear multiple times on the
same sequence, leading to the difference between these two numbers.

Finally, we group sequences from the same organism according to the
NCBI
code that is assigned by
UniProt,
allowing us to count the number of distinct sequences on which the
domain is found. This value is shown in the
pink boxes.

We use the NCBI species tree to group organisms according to their
taxonomy and this forms the structure of the displayed tree.
Note that in some cases the trees are too large (have
too many nodes) to allow us to build an interactive tree, but in most
cases you can still view the tree in a plain text, non-interactive
representation. Those species which are represented in the seed
alignment for this domain are
highlighted.

You can use the tree controls to manipulate how the interactive tree
is displayed:

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highlight species that are represented in the seed alignment

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Structures

For those sequences which have a structure in the
Protein DataBank, we
use the mapping between UniProt, PDB and Pfam coordinate
systems from the PDBe group, to allow us to map
Pfam domains onto UniProt sequences and three-dimensional protein
structures. The table below
shows the structures on which the ShK
domain has been found. There are 5
instances of this domain found in the PDB. Note that there may be
multiple copies of the domain in a single PDB structure, since many
structures contain multiple copies of the same protein sequence.